This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Core 1 focuses on developments associated protein localization within the cell, the protein-protein interactions, the quantitative dynamics and protein trafficking, and the modification states of proteins applying high throughput proteomics measurements. For this annual report top-down and bottom-up proteomics have been applied to characterize the post-translational modifications (PTMs) of protein isoforms with the initial emphasis being on protein phosphorylation. Measurement of the phosphoproteome included measuring the low abundance phosphotyrosine modifications in addition to the more abundant pSer and pThr modifications. To facilitate PTM characterization in intact proteins the Center has focused on developing an enhanced version of RePlay proteomics. Advanced methods of sample preparation, pressure digestion and sonication, have been developed in support of measuring differential protein abundances for systems biology studies. Reported for the first time is the area of activity-based proteomics (ABP) that represents a new direction for the Center in Core 1 providing a more targeted approach to probing the sub-proteomes of tissues, cells, and biofluids. Methods in subcellular fractionation are being implemented in the Center to achieve Core 1 goals for characterizing proteins in cells.